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Functional Characterization of the TRP-Type Channel PKD2L1

  • Author / Creator
    Hussein, Shaimaa A
  • Polycystic kidney disease (PKD) protein 2 Like 1 (PKD2L1), also called transient receptor potential polycystin-3 (TRPP3), regulates Ca2+-dependent hedgehog signalling in primary cilia, intestinal development and sour tasting but with an unclear mechanism. PKD2L1 is a Ca2+-permeable cation channel that is activated by extracellular Ca2+ (on-response) in Xenopus oocytes. PKD2L1, co-expressed with PKD protein 1 Like 3 (PKD1L3) receptor protein, exhibits acid-induced off-response activation (i.e., activation occurs only after the removal of an acidic solution). Whether PKD1L3 participates in acid sensing and what is the exact mechanism of PKD2L1 channel function remain unclear. In Chapter 2, we provided an answer for the first question. By using the two-microelectrode voltage-clamp, site directed mutagenesis, Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence, we showed that PKD2L1 expressed alone in oocytes exhibits sustained off-response currents in the absence of PKD1L3. PKD1L3 co-expression augmented the PKD2L1 plasma membrane localization but did not alter the observed properties of the off-response. PKD2L1 off-response was inhibited by an increase in intracellular Ca2+. We also identified two intra-membrane residues aspartic acid 349 (D349) and glutamic acid 356 (E356) in the third transmembrane domain that are critical for PKD2L1 channel function. Our study suggests that PKD2L1 may itself sense acids and defines off-response properties in the absence of PKD1L3. It was previously reported that PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. Thus, it remains unclear how PKD2L1 proteins oligomerize into a functional channel. In Chapter 3, by use of SDS-PAGE, blue native PAGE and mutagenesis we identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization and the channel function of PKD2L1 and PKD2L1/PKD1L3. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggests that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process. In Chapter 4, we studied roles of phospholipase C (PLC), lipid messengers and phosphorylation in PKD2L1 channel function. As PKD2L1 is expressed in type III taste receptor cells (TRCs) which are known to have a phospholipase C-dependent Ca2+ signalling, we wondered whether PKD2L1 function is downstream of a PLC pathway or is affected by different lipid messengers. Using Xenopus oocyte expression system, two-microelectrode voltage-clamp (TMVC) and immunofluorescence (IF), we found that PKD2L1 function is indeed downstream of a PLC pathway. PLC activator m-3M3FBS opens the channel in a Ca2+-dependent manner, while the PLC inhibitor U73122 inhibits the Ca2+-induced channel function. Moreover, we found that phorbol 12-myristate 13-acetate (PMA), a commonly used PKC activator, but not its biologically non-active analogue, 4α-phorbol 12,13-didecanoate (4αPDD), inhibits PKD2L1 channel function and reduces its plasma membrane localization. Consistently, when we used a PKC blocker GF109203x the channel activity was increased. Further, we tested a number of potential phosphorylation sites and found that PKD2L1 mutant T338A has a substantially reduced response to the PMA treatment. Our results indicate that 1) PLC regulates PKD2L1 channel function and that PKD2L1 inactivation is PKC-dependent, and 2) PKC regulates PKD2L1 channel function through phosphorylating threonine 338 and affecting its surface membrane density. This study will constitute an important step towards understanding the molecular mechanism underling the biological functions of PKD2L1 such as its role in sour tasting. In summary, our studies constitute valuable contributions to understanding the function and regulation of PKD2L1.

  • Subjects / Keywords
  • Graduation date
    Fall 2015
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3P84495F
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Gallin, Warren (Biological Sciences)
    • Lytton, Jonathan (Biochemistry & Molecular Biology)
    • Leslie, Elaine (Physiology)
    • Alexander, Todd (Physiology)